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Previous Article | Next Article 
Infection and Immunity, January 2007, p. 193-200, Vol. 75, No. 1
0019-9567/07/$08.00+0 doi:10.1128/IAI.01148-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vß1+ Jß1.1+/V
2+ J49+ CD4+ T Cells Mediate Resistance against Infection with Blastomyces dermatitidis
Marcel Wüthrich,1*
Hanna I. Filutowicz,1
Holly L. Allen,2
George S. Deepe,2 and
Bruce S. Klein1,3,4,5
Departments of Pediatrics,1
Internal Medicine,3
Medical Microbiology and Immunology,4
Comprehensive Cancer Center, University of Wisconsin Medical School, University of Wisconsin Hospital and Clinics, Madison, Wisconsin 53792,5
Division of Infectious Diseases, University of Cincinnati College of Medicine and Veterans Affairs Hospital, Cincinnati, Ohio 452672
Received 21 July 2006/
Returned for modification 16 September 2006/
Accepted 30 September 2006
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ABSTRACT
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Immunization with a cell wall/membrane (CW/M) and yeast cytosol extract (YCE) crude antigen from Blastomyces dermatitidis confers T-cell-mediated resistance against lethal experimental infection in mice. We isolated and characterized T cells that recognize components of these protective antigens and mediate protection. CD4+ T-cell clones elicited with CW/M antigen adoptively transferred protective immunity when they expressed a V
2+ J49+/Vß1+ Jß1.1+ heterodimeric T-cell receptor (TCR) and produced high levels of gamma interferon (IFN-
). In contrast, Vß8.1/8.2+ CD4+ T-cell clones that were reactive against CW/M and YCE antigens and produced little or no IFN- either failed to mediate protection or exacerbated the infection depending on the level of interleukin-5 expression. Thus, the outgrowth of protective T-cell clones against immunodominant antigens of B. dermatitidis is biased by a combination of the TCR repertoire and Th1 cytokine production.
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INTRODUCTION
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Blastomycosis is one of the principal endemic systemic mycoses of North America. The disease is caused by the thermal dimorphic fungus Blastomyces dermatitidis, which exists in the mycelial form in nature and converts to a yeast form at 37°C in mammalian hosts. Infection with B. dermatitidis is usually acquired by the inhalation of aerosolized spores, which are deposited in the alveolus, convert, and then produce an asymptomatic infection or a mild or progressive pneumonia. Blastomycosis can produce a fatal infection if it goes undiagnosed or presents as a fulminant, progressive pneumonia (14). Most infections occur in immunocompetent hosts, and yet B. dermatitidis can also produce an opportunistic infection in immunocompromised hosts (11).
A vaccine against B. dermatitidis would be clinically useful for the prevention of infection, but a commercial vaccine is not currently available. Recently, a genetically engineered and attenuated strain of B. dermatitidis has been created and shown to be an effective live attenuated vaccine in a murine model of lethal pulmonary infection (17). Animals that received injections twice, given 2 weeks apart, all survived, and most acquired sterilizing immunity after a lethal pulmonary challenge with a wild-type virulent yeast strain of B. dermatitidis (17). The vaccine induces delayed-type hypersensitivity and polarized type I cytokine responses, which are linked with resistance. Crude antigen preparations from a cell wall/membrane extract (CW/M) and a yeast-cytosol extract (YCE) of the vaccine strain also induced polarized and protective immune responses in mice (17). Vaccine-induced immunity to B. dermatitidis is critically dependent on the presence of thymus-derived lymphocytes (either CD4+ or CD8+ T cells), and the contribution of the different subsets depends on the underlying immune competency of the host (16, 18-21). For example, in competent hosts, CD4+ T cells play the major role in vaccine immunity, and their action is critically dependent on the production of the type I cytokines gamma interferon (IFN-), tumor necrosis factor alpha (TNF-), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (19). In immunosuppressed hosts lacking CD4+ T cells, however, CD8 T cells are able to compensate and mediate protective immunity (18). The antigen-specific T cells that drive vaccine-induced protective immune responses to this fungus and the chief antigens that drive the responses remain unknown.
In the present study, we sought to isolate and characterize antigen-specific T cells that mediate resistance to B. dermatitidis after vaccination. Our goals were to (i) generate T-cell lines and clones against the CW/M and YCE crude protective antigens derived from the attenuated vaccine strain of B. dermatitidis; (ii) characterize these lines and clones phenotypically and functionally with regard to their T-cell subset, cytokine production, and capacity to mediate protective immunity upon adoptive transfer; and (iii) delineate the V and Vß repertoire of T-cell clones that drive the protective immune responses against this agent. Our results indicate a bias in the T-cell receptor (TCR) repertoire toward Vß1 for protective T-cell clones and Vß8.1/8.2 for nonprotective and disease-enhancing clones.
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MATERIALS AND METHODS
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Fungi and growth conditions.
The strains used were ATCC 26199 (9), a wild-type virulent strain, and the isogenic, attenuated mutant lacking BAD1, designated strain 55 (2). Isolates of B. dermatitidis were maintained as yeast on Middlebrook 7H10 agar with oleic acid-albumin complex (Sigma Chemical Co., St. Louis, MO) at 39°C.
Mouse strains.
Inbred strains of C57BL/6 and IL-4-deficient B6.129P2-IL-4tm1Cgn/J stock 2253 mice (10) were obtained from Jackson laboratories, Bar Harbor, ME. Male mice were 7 to 8 weeks of age at the time of these experiments. Mice were housed and cared for according to guidelines of the University of Wisconsin Animal Care Committee, which approved all aspects of this work.
Antibodies and other reagents.
Interleukin-4 (IL-4) monoclonal antibody (MAb) (a gift from Craig Reynolds, Biological Resources Branch, Frederick, MD) and 600 U of murine recombinant IFN- (rIFN-; Pharmingen, San Diego, CA)/ml were used as previously described (17).
Preparation of crude antigens of recombinant strain 55.
Antigens were extracted from yeast cells of strain 55 according to the method of Gomez et al. (6) as previously described elsewhere (17). Briefly, yeast cells were grown in liquid histoplasma macrophage medium for 5 days. Harvested cells were killed in thimerosal (1:10,000 [wt/vol]) in phosphate-buffered saline (PBS; pH 7.2) for 1 h at 37°C, followed by overnight incubation at 4°C. Washed, pelleted cells were suspended in PBS containing 1 mM phenylmethylsulfonyl fluoride, 5 µM leupeptin, and 5 x 10–4 M sodium EDTA. Cells were disrupted (Braun Biotech, Allentown, PA) at 4°C for 2 min using alternating 30-s cycles of homogenization and cooling. Disrupted cells were centrifuged, and the supernatant was saved and termed YCE. Pelleted cell walls were extracted with urea (6). Supernatant was saved and termed CW/M. Antigen preparations were dialyzed against PBS and centrifuged at 31,000 x g for 30 min. Soluble material was sterilized with a 0.2-µm-pore-size filter and stored at –20°C.
Antigen-specific T-cell lines and clones.
C57BL/6 mice were immunized subcutaneously with yeast strain 55 given in two locations, dorsally and at the base of the tail, three times, two weeks apart. At 2 weeks after immunization, mice received 200 µg of CW/M or YCE emulsified in complete Freund adjuvant administered into the base of the tail and both footpads. After 10 days, the draining lymph node cells were removed and cultured in complete Dulbecco modified Eagle medium (containing 50 U of human recombinant IL-2/ml, 10% fetal bovine serum, 16 mM HEPES buffer, 2 mM L-glutamine, 100 U of penicillin/ml, 100 µg of streptomycin/ml, 0.12 mg of L-arginine/ml, 0.035 mg of L-asparagine/ml, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 0.6 mg of folic acid/ml, and 5.5 x 10–5 M 2-mercaptoethanol) at a concentration of 2 x 106 cells/ml with 6 x 106 syngeneic, irradiated (1,800 R) spleen cells/ml as antigen-presenting cells and 12.5 µg of CW/M or YCE antigen/ml. Stimulated cells were harvested 8 days later and cultured at 2 x 105 cells/ml in the presence of antigen and 2 x 106 antigen-presenting cells/ml in a 24-well plate. To maintain these T-cell lines, cells were restimulated every 2 weeks with fresh antigen and antigen-presenting cells.
To generate clones, T cells from these lines were plated at a limiting dilution (30, 10, and 3 cells/96-well) with 8 x 105 irradiated splenocytes in the presence of 0.5 ng of recombinant, murine IFN- (R&D Systems, Minneapolis, MN)/ml and 12.5 µg of antigen/ml in complete Dulbecco modified Eagle medium containing 50 U of human recombinant IL-2/ml as detailed above. After 7 days of culture, the wells were examined microscopically for the emergence of single clones. One and three weeks later, expanded clones were transferred to 48- and 24-well plates, respectively. T-cell clones were maintained in 24-well plates at 2 x 105 cells/ml and restimulated every other week with 2 x 106 freshly irradiated, syngeneic splenocytes, 12.5 µg of antigen/ml, and 50 U of human recombinant IL-2/ml.
Cytokine protein measurements.
Cell culture supernatants were generated in 24-well plates in 1 ml containing 2 x 106 irradiated splenocytes, 2 x 105 T cells, and 5 µg of concanavalin A/ml or 12.5 µg of Blastomyces CW/M or YCE antigen/ml (17). CW/M antigen contained <0.1 endotoxin unit/ml. Supernatants were collected after 96 h of coculture. IFN-, TNF-, GM-CSF, IL-4, IL-5, IL-10, and transforming growth factor ß1 (R&D Systems) were measured by enzyme-linked immunosorbent assay according to manufacturer's specifications (the detection limits were 0.05, 0.02, 0.05, 0.05, 0.04, 0.05, and 0.05 ng/ml, respectively).
TCR analysis by flow cytometry.
To determine the identity of the V and Vß chains on the surface of T-cell clones, aliquots of each clone containing 105 cells were incubated with one of the biotinylated antibodies to Vß2, -3, -4, -5.1/5.2, -6, -7, -8.1/8.2, -8.3, -9, -10, -11, -12, -13, and -14 and V2 for 20 min at 4°C. After two washes, the cells were incubated with streptavidin-phycoerythrin for 20 min at 4°C, washed twice, and fixed with 1% paraformaldehyde. Fluorescence was measured using the LSRII flow cytometer.
Analysis of TCR- and -ß genes.
T cells (2 x 106) of individual T-cell clones were harvested, and total RNA was isolated using the RNeasy minikit (QIAGEN) as described previously (18). A total of 0.5 to 1 µg of RNA in a final volume of 20 µl was reverse transcribed using random hexamers (2.5 µM), oligo(dT) primers (2.5 µM), and the TaqMan RT-PCR kit (Applied Biosystems). Aliquots of 1 µl of the reverse transcription reaction were used as a template in a parallel PCR. Vß gene amplification and sequencing was performed with all four CW/M clones and all nine YCE clones. V gene amplification was done only for the four CW/M clones, since we knew the identity of the V domain (V2) of these CW/M clones from flow cytometry analysis, but we lacked this information for the YCE clones. Each tube contained a common antisense primer specific to the constant region of the ß-chain and each of the 20 Vß-specific sense primers (3). Amplification of the TCR alpha gene was performed with an antisense primer specific to the constant region of the -chain and the V2-specific sense primer (3). PCR was performed in 25-µl reaction mixtures with 1.25 U of Taq polymerase (Invitrogen), 1x PCR buffer (20 mM Tris-HCl and 50 mM KCl), 1.5 mM MgCl2, 200 µM concentrations of each deoxynucleoside triphosphate (Promega), and a final primer concentration of 1 µM. Reaction mixtures were denatured at 94°C for 45 s, annealed at 59°C for 45 s, and extended at 72°C for 45 s in a GeneAmp PCR system 9700 Thermocycler (Applied Biosystems). The resulting PCR products were cloned into the PCR2.1 vector by using the TOPO-TA cloning kit (Invitrogen) according to the manufacturer's instructions. Random colonies were picked for each clone and submitted for automated sequencing at the University of Cincinnati DNA core facility. The sequence was analyzed by using DNAMan V5.2.9 (Lynnon Biosoft).
Adoptive transfer of T cells in vivo and experimental infection.
T-cell lines and clones were transferred intravenously into irradiated (5.5 Gy) wild-type C57BL/6 recipient mice. Irradiated mice were rested for up to 6 weeks before infection. Mice were infected intratracheally with 2 x 102 to 2 x 103 wild-type strain 26199 yeast cells as described previously (17). Infected mice were monitored for survival or analyzed 3 weeks after infection for the extent of lung infection, determined by plating homogenized lung and the enumeration of yeast CFU on brain heart infusion (Difco, Detroit, MI) agar.
Statistical analysis.
Differences in number of CFU were analyzed by using the Wilcoxon rank test for nonparametric data (5). A P value of <0.05 is considered statistically significant.
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RESULTS
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Generation and in vitro characteristics of T-cell lines.
As described previously (17), two crude antigen preparations were derived from B. dermatitidis yeast cells: a CW/M and a YCE. Both of the antigens, when administered subcutaneously in adjuvant to mice twice (2 weeks apart), significantly protected vaccine recipients against experimental infection, as measured by the reduction in lung CFU after a lethal pulmonary infection with the virulent wild-type strain. This resistance is mediated by ß CD4+T cells in immunocompetent hosts (17, 19). We sought to identify, isolate, and characterize features of these antigen-specific T cells.
T-cell lines raised against CW/M and YCE antigens proliferated vigorously in response to their respective antigens (Fig. 1A). The two lines were analyzed for the type and amount of cytokines they produced in response to these antigens. The T-cell line initially raised with CW/M produced a polarized type II cytokine profile (IL-4 and IL-5), whereas the line generated with YCE showed a mixed cytokine phenotype, by producing the type I cytokines IFN-, TNF-, and GM-CSF and also the type II cytokines IL-4 and IL-10 (Fig. 1B). Based on their cytokine profiles, we termed these T-cell lines CW/M Th2 and YCE Th1/2. Since type II cytokines are thought to antagonize immunity to fungi (13), we generated additional T-cell lines against both antigens under conditions that favor a Th1 phenotype as described in Materials and Methods.
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FIG. 1. CW/M and YCE T-cell lines proliferate and produce cytokines in response to crude antigens of B. dermatitidis. T-cell lines raised against CW/M and YCE antigen were cocultured with medium alone or CW/M or YCE antigen (12.5 µg/ml). The data are mean values of three independent experiments. (A) Proliferation was measured as the cpm of [3H]TdR incorporated into proliferating cells after 4 days of coculture. The cpm values for medium control-stimulated T cells were from 300 to 1,500 cpm and were subtracted. (B) Cytokines were measured in the supernatants of T cells, cultured as described for panel A. The cytokine concentrations of medium control-stimulated T cells for all cytokines measured were between 0.001 and 0.1 ng/ml and were subtracted. +, endpoint not determined; i.e., the final concentration of IFN- was not measured by an endpoint titration.
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To generate T-cell lines with a polarized type I cytokine response, we took two approaches. For the generation of a CW/M-Th1 line, we cultured draining lymph node cells from mice vaccinated as described above in medium supplemented with rat anti-mouse IL-4 MAb (1 µg/ml) and murine rIFN- (600 U/ml) as described previously (17). The resulting line released IFN-, TNF-, and GM-CSF, which have been linked with T-cell-mediated resistance to B. dermatitidis (17, 19-21), but none of the type II cytokines IL-4, IL-5, and IL-10 (Fig. 1B). After eight rounds of stimulation, the CW/M-Th1 line maintained its phenotype without the addition of rIFN- and anti-IL-4 MAb. For the generation of a YCE-Th1 line, we vaccinated IL-4-deficient mice (10) as described above and stimulated draining lymph node cells with YCE antigen without any further type I cytokine-favoring modifications. The YCE-Th1 T-cell line produced only type I and no type II cytokines upon stimulation with antigen (Fig. 1B).
T cells from all four lines were analyzed phenotypically by fluorescence-activated cell sorting (FACS) to determine their subset; all of them stained positively for CD3 and CD4 markers but did not express the CD8 marker, as might be expected since they were raised in vitro with soluble antigens (data not shown). Of interest, while the lines responded to the antigen with which they were raised, they also responded to the other antigen; e.g., the CW/M Th1 line responded to YCE antigen and vice versa (Fig. 2A). This finding suggests that there is sharing of the major T-cell antigen(s) in the two preparations.
Functional properties of T-cell lines in the adoptive transfer of protective immunity.
Since a goal of the present study is to generate and characterize T-cell clones that recognize individual antigen components that are responsible for eliciting protective T-cell responses, we sought to identify T-cell lines that are functionally protective in vivo. To evaluate this function of the T cells, we allowed them to repopulate mice that had been irradiated sublethally before T-cell transfer. After rest and repopulation over 6 weeks, mice that received T cells and controls were challenged with a lethal dose of B. dermatitidis. The functional capacity of the T-cell lines in vivo correlated with their cytokine profile in vitro. Th1 lines that were raised against CW/M and YCE antigens successfully transferred resistance, as defined by a significant reduction in lung CFU (Fig. 3). The YCE Th1/2 and CW/M Th2 T-cell lines exacerbated infection, and this was associated with the elaboration of the type II cytokines IL-4, IL-5, and IL-10. Based on the in vivo functional data, we cloned Th1 cell lines to characterize protective T cells at the clonal level.
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FIG. 3. Burden of lung infection after adoptive transfer of T-cell lines. Six weeks after the transfer of T cells (2.5 x 107 cells/mouse), mice were infected intratracheally with 4 x 102 yeast cells of wild-type strain 26199. Controls received mock transfer with PBS. At 3 weeks postinfection, mice were analyzed for lung infection. Median values (middle bar), 50th percentile values (boxes), and minimum (lowest bar) and maximum (top bar) values are shown. *, P < 0.0008; **, P < 0.33 (versus the control group).
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Generation and in vitro characterization of T-cell clones.
We generated and characterized four T-cell clones from the CW/M Th1 cell line and nine T-cell clones from the YCE Th1 cell line. Each group of clones responded to the eliciting antigen and also responded to various degrees to the reciprocal antigen (Fig. 2B), reinforcing the idea that the two crude antigens share major T-cell antigens.
The CW/M clones proliferated (Fig. 4A) and produced type I cytokines IFN-, TNF-, and GM-CSF in response to CW/M (Fig. 4B). T-cell clones 5, 6, and 7 produced substantial amounts of IFN- upon endpoint titration of the amount of cytokine produced (85, 110, and 140 ng/ml, respectively) but no detectable amounts of IL-4, IL-5, IL-10, or transforming growth factor ß (Fig. 4B and data not shown). In contrast, T-cell clone 1 produced a significantly smaller amount of IFN- (1 ng/ml) but concomitantly produced a high level of IL-5 (7.9 ng/ml).
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FIG. 4. Proliferation and cytokines by T-cell clones. (A) Proliferation by CW/M clones and CW/M Th1 T-cell line (as a reference) in vitro in response to stimulation with CW/M antigen. cpm values for medium control-stimulated T cells were from 63 to 230 cpm and were subtracted. The production of IFN-, TNF-, GM-CSF, and IL-5 by CW/M (B) and YCE (C) T-cell clones was assayed as described in Fig. 2 and 3. Panels A and B represent an average of three independent experiments. The cytokine concentrations of medium control-stimulated T cells for all cytokines measured were from 0.001 to 0.1 ng/ml and were subtracted. +, these bars do not represent endpoint values. To determine the exact concentrations of cytokines, we performed endpoint titrations in one representative experiment. The CW/M line and clones 5, 6, and 7 produced 45, 85, 110, and 140 ng of IFN-/ml, respectively, and clone 1 secreted 7.9 ng of IL-5/ml.
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The YCE T-cell clones showed a different cytokine pattern (Fig. 4C). For example, all clones exhibited a polarized type I cytokine phenotype, producing type I cytokines TNF- and GM-CSF, and they failed to produce type II cytokines IL-4, IL-5, and IL-10. Only T-cell clones 8, 11, and 26 produced IFN- in response to antigen stimulation, but the amount produced was only minimal (0.27 ± 0.02, 0.92 ± 0.05, and 0.227 ± 0.009 ng/ml, respectively) compared to the amounts produced by the CW/M T-cell clones.
T-cell clones adoptively transfer resistance.
To evaluate whether and which CW/M and YCE T-cell clones are functionally protective in vivo, we adoptively transferred them into sublethally irradiated mice as described above and let the T cells expand over a period of 6 weeks before animals were challenged with a lethal dose of B. dermatitidis. Three of the four T-cell clones (clones 5, 6, and 7) raised against CW/M transferred significant protective immunity to the infection, as defined by a significant reduction in lung CFU. In sharp contrast, clone 1 exacerbated infection (Fig. 5A). Hence, the ability of CW/M T-cell clones to transfer protection in vivo was functionally linked with the production of high levels of IFN- and the absence or low levels of type II cytokines, in particular IL-5, in vitro. Conversely, the ability to enhance infection, as in the case of T-cell clone 1, was functionally linked with a reciprocal pattern of cytokine production, namely, a high level of IL-5 relative to type I cytokine, especially IFN-.
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FIG. 5. T-cell clones transfer protective immunity. CW/M (A) and YCE (B and C) clones and the respective founder T-cell line (2 x 107 T cells/mouse) were adoptively transferred into sublethally irradiated wild-type C57BL/6 recipients who were then allowed to rest for 6 months. Mice were infected with 2 x 102 wild-type yeast cells intratracheally, and lung CFU were analyzed 3 weeks later. *, P < 0.003 versus controls with no cell transfer. Panel A is representative of three independent experiments; panels B and C are representative of two independent experiments. Median values (middle bar), 50th percentile values (boxes), and minimum (lowest bar) and maximum (top bar) values are shown. The numbers above the bars indicate the n-fold reductions in numbers of lung CFU versus the number of lung CFU after mock transfer with PBS.
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Of the nine YCE T-cell clones, only one (clone 11) reduced the lung CFU count significantly compared to mice that received no T cells (Fig. 5B and C). Clone 11 produced the highest level of IFN- in vitro (0.92 ± 0.05 ng/ml) among all of the YCE clones.
Thus, we have generated Blastomyces antigen-specific T-cell clones with defined cytokine phenotypes in vitro and functional resistance in vivo, four of them protective and one enhancing upon infection.
TCR gene usage is associated with resistance.
To investigate whether the protective capacity of the T-cell clones in vivo is functionally linked with TCR usage, we sequenced the Vß genes of the CW/M and YCE T-cell clones. Remarkably, the three most protective clones of the present study, CW/M clones 5, 6, and 7, showed the same TCR-ß usage. Each of them utilized Vß1 and Jß1.1, and the amino acid motif of the CDR3 region was uniformly SQEGTGG (Table 1) . In contrast, the disease-enhancing CW/M clone (T-cell clone 1) and all of the YCE T-cell clones showed a different TCR-ß usage. Each of them instead utilized Vß8.1/8.2 and Jß2.6, and the amino acid motif of the CDR3 region was GTGGV. The identities of the Vß families were independently confirmed by surface staining of the T-cell receptors and analysis by flow cytometry (Table 1). FACS analysis also showed that all four CW/M clones were V2 positive, whereas all of the YCE clones failed to stain with this antibody and thus were not. PCR amplification with a V2-specific sense primer and a C antisense primer (3) and subsequent sequence analysis revealed the J identity and CDR3 domain for clones 5, 6, and 7 (J49 and AGGYQNFYFGKGSLTVIP, respectively) and clone 1 (J34 and VPNTNKVVFGTGTRLQVLP, respectively). These data indicate that all three protective clones (5, 6, and 7) express identical TCR-ß heterodimers that are distinct from those of the disease-enhancing T-cell clone and also from other clones in the present study.
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DISCUSSION
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In this study, we generated and characterized T-cell lines and clones that are specific for B. dermatitidis determinants in two crude antigen preparations and that influence disease resistance in vivo. Four T-cell clones conferred protection—three of four CW/M clones and one of nine YCE clones—and one clone (CW/M clone 1) enhanced infection. To our surprise, only two Vß families were detected among the 13 Blastomyces-reactive clones analyzed given the fact that the crude antigen extracts harbor multiple proteins (17). Moreover, we have uncovered a strong bias in the TCR repertoire of protective and nonprotective clones. Vß1+ clones were uniformly protective, whereas Vß8.1/8.2+ clones were chiefly nonprotective or disease enhancing. It is not clear whether the limited representation of only two Vß families among all 13 clones described in the present study was a consequence of the preferential expansion of Vß1+ and Vß8.1/8.2+ T cells during the vaccination in mice or a result of a biased presentation of particular peptides during the in vitro expansion of the T-cell lines and clones.
There is precedence in the literature for preferential expansion of a particular Vß family during experimental infection or vaccination with other infectious diseases. During primary and secondary infections with the fungal pathogen Histoplasma capsulatum, consistent oligoclonal expansion of Vß4+ and Vß6+ T cells was observed (7, 8). Vß4+ T cells expanded in the lungs of mice during the peak and early clearance phases of primary infection (7). This expansion of Vß4+ T cells was essential for optimal clearance of the fungus from tissues. Primary infection with a nonlethal dose of H. capsulatum yeast that protects against a secondary pulmonary infection with a lethal dose resembles the antigen complexity of subcutaneous vaccination with live attenuated B. dermatitidis yeast and subsequent priming with the complex crude CW/M and YCE antigen preparations that was used here to generate protective T-cell lines and clones. Reinfection histoplasmosis significantly increased the number of Vß6+ T cells during the peak and early resolution phases of infection (8). Depletion of Vß6+ T cells but not Vß4+ T cells induced a modest but significant delay in fungal clearance. The simultaneous depletion of Vß4+ and Vß6+ T cells induced a more pronounced impairment of host resistance. These results indicate that primary and secondary infection with H. capsulatum evoked a distinct bias in the TCR repertoire, illustrating the dynamic interactions between Vß families in response to a microbial encounter. In addition, vaccination of mice with the protective antigen heat shock protein 60 of H. capsulatum or its immunodominant F3 fragment also induced a highly restricted Vß T-cell response involving Vß 8.1/8.2 and Vß6 T cells, respectively (4, 15). An analysis of the T-cell receptor repertoire used by mice infected with Leishmania major revealed the expansion of a restricted population of CD4+ Vß4+ cells in both progressive infection and protective immunity and across several major histocompatibility haplotypes (12).
Sequence analysis of the expressed TCR-ß unit indicated that within each Vß family the Jß usage and CDR 3 expression were the same. Protective Vß1+ clones expressed the Jß1.1+ element and the CDR3 motif SQEGTGG, whereas the chiefly nonprotective or disease-enhancing Vß8.1/8.2+ clones expressed the Jß2.6+ element and the CDR3 motif GTGGV. The outgrowth of a very limited set of Vß families in conjunction with a preferential usage of Jß families and CDR3 expression suggests that the T-cell response against these repetitively administered (in vivo and in vitro) crude antigens is driven by few proteins and/or peptides. The identification of these proteins is currently under investigation. Convergence toward a small number of dominant CDR3 motifs has been reported during experimental infection with H. capsulatum. Before inoculation and during the early phase of infection, the CDR3 sequences of dominating Vß4+ and Vß6+ T cells in the lung tended to be diverse but then converged to oligoclonality during the peak and early resolution phases of secondary infection (8). Hence, it is possible that a dominant antigen or antigens in a complex crude antigen preparation such as CW/M and YCE could lead to a strong bias in the Vß repertoire of outgrowing T-cell clones when administered repetitively during vaccination and in vitro stimulation, as has been reported for reinfection histoplasmosis.
The most interesting clones generated in the present study are the CW/M-reactive clones since they all showed a clear phenotype in adoptive-transfer experiments. Clones 5, 6, and 7 were uniformly protective, whereas clone 1 was consistently disease enhancing. We therefore sought to characterize the -unit of the TCR of those clones. FACS and sequence analysis showed that the three protective clones express identical TCR- units; thus, all three clones have the same heterodimeric TCR, indicating that they are sibling clones. Although the TCR- unit of clone 1 was V2 positive like the other CW/M clones, the J element or CDR3 region and the TCR-ß were different, indicating that clone 1 and clones 5, 6, and 7 must recognize different epitopes. Even though clone 1 shares the TCR-ß unit with the YCE clones, it expresses a different TCR- and will likely exhibit a distinct antigen specificity. Since all nine YCE clones have the same TCR-ß, it is plausible that they are siblings clones, but without having sequenced the TCR- we do not know this with certainty. Hence, the 13 clones generated and characterized in the present study recognize at least three or more different epitopes.
When the T-cell lines and clones were analyzed for cytokine production, we observed a correlation between the production of IFN- and IL-5 and protective efficacy. High levels of IFN- and no or low levels of IL-5 augmented resistance and vice versa. For example, CW/M Th2 and YCE Th1/2 lines and CW/M clone 1 that expressed a Th2 or mixed Th1/2 cytokine profile were not protective or disease enhancing. On the other hand, the CW/M Th1 lines and CW/M clones 5, 6, and 7 produced exceptionally high levels (
50 ng/ml) of IFN- but no or low levels of IL-5 (clone 6) and adoptively transferred high levels of resistance. In the complete absence of type II cytokines, as in the case of the YCE Th1 line and YCE clone 11, even low levels of IFN- (
1 ng/ml) were associated with reduced lung CFU. The level of IFN- produced by YCE clone 11 was the only parameter measured that correlated with resistance compared to the other Vß8.1/8.2+ YCE clones, which were nonprotective. However, we cannot exclude the possibility that other cytokines not measured in the present study determined the activity of YCE clone 11 and other T-cell clones as well. Alternatively, YCE clone 11 could express a TCR- chain and a CDR3 region that is distinct from the other Vß8.1/8.2+ YCE clones, resulting in differential antigen specificity and protection. Hence, our data suggest that the ability of a particular T-cell clone to resist infection with B. dermatitidis is likely to be a consequence of both antigen specificity and the cytokine profile.
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ACKNOWLEDGMENTS
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This study was supported by NIH grants AI-40996 and AI-61298 to B.S.K. and AI-42747 and AI-34361 to G.S.D. from the U.S. Public Health Service.
We thank Tom S. Sullivan from the Department of Pediatrics for help with sequence analysis on the TCR- sequences and Jens Eickhoff from the Department of Biostatistics and Medical Informatics for assistance with statistical analysis.
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FOOTNOTES
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* Corresponding author. Mailing address: University of Wisconsin Hospitals and Clinics, 600 Highland Ave., K4/444, Madison, WI 53792. Phone: (608) 263-6203. Fax: (608) 263-0722. E-mail: mwuethri{at}wisc.edu. 
Published ahead of print on 9 October 2006.
Editor: A. Casadevall
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Infection and Immunity, January 2007, p. 193-200, Vol. 75, No. 1
0019-9567/07/$08.00+0 doi:10.1128/IAI.01148-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
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ABSTRACT
INTRODUCTION
